JPH06500555A - Peptide drugs for disease treatment - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Peptide drugs for disease treatment The present invention is in the field of biology/biochemistry and in particular anti-inflammatory prophylactic or therapeutic agents. having a defined amino acid sequence that is a useful drug when used as a therapeutic agent Related to peptides.

During an inflammatory response, peripheral blood leukocytes consist of neutrophils and monocytes, endothelial cells binds to and moves through the layer, and moves across the basement membrane in response to chemotactic factors. cross and enter the infected tissue, where leukocytes suppress the infecting organism. or effective in removal. When the host's defense system responds appropriately to infection The inflammatory response occurs when white blood cells enter only the infected area, eventually damaging healthy tissue. closely suppressed to prevent it from happening. In certain conditions, especially sepsis, white The action of blood cells is not tightly suppressed and eventually oxygen-induced free radicals as well as neutrophils Extensive vascular damage as a result of release of proteases and phospholipases and can thus cause significant cell and tissue damage.

Harlan, J, Gate 9.1987, Act Med 5cand Su 1 ．． ．． 715: 123; Weiss, S., 1989, "Schiff" - Mu and L Yu I Eng, T.: 365° For example, sepsis-associated neutrophil-mediated endothelial damage , has been implicated in loss of vascular integrity, thrombosis, and tissue necrosis.

An early event leading to neutrophil damage to endothelial cells is the attachment of neutrophils to the endothelial cell surface. be. In a significant part, this causes neutrophils to bind to the endothelial cell surface. Mediated by associated cell adhesion molecules. The neutrophil adhesion molecule is ICAM-1 ( It binds to molecules on the surface of endothelial cells called intercellular adhesion molecules. To this day, this reaction A partial list of adhesion molecules identified as being associated with lymphocyte-associated antigen-1 (L FA-1), macrophage antigen-1 (MAC-1”), also phylum 0-1, OK M-1, and complementary reporter type 3, and p, 1so, 95, and also complementary reporter type 3. complementary reporter type 4 (CR-4) and LeuM-5. These molecules are Specifically, the LFA-1 family, leukocyte adhesion protein, IeuCAM, and leukocyte protein It has been called tegrin. All three molecules are α-β heterodimers. Ru. The β subunits are the same in all three molecules, but the α subunits are different. . Kurzinger, T.A., and Springer, T.A., 1982, J. , of Biol, Chem,, 257: 1241: 5anct+ez -Madrid, F., et al., 1983, Lhl1E, 158: 1785; Trowbrige, 1. S., and Omary,,M.

B1.1981, Beard Price Needle Problem-278: 3039° Integration of 3 White Blood Cells Phosphorus is primarily expressed by immune cells. For example, LFA-1 virtually all present on immune cells.

Xurzinger, , and Springer, T.A., supra. l Ac-1 is associated with monocytes, macrophages, granulocytes, large granular lymphocytes, and immature is expressed by CD-5+BIII cells. De La Hera, A, et al. 1988, Eur, J. of IIl Sunol, 18: 1131. p1 The 50.95 protein shares the same cell type distribution as MAC-1, but upon activation It is further expressed by lymphocytes, as well as hairy cell leukemia. that is a marker for the latter disease. Schwarting, R. et al., 1985, Blood, 65: 974; Miller, B.

A. et al., 1985, J. of Is+s+unoL 134: 3286° Lab. Studies have implicated leukocyte integrins in cell adhesion events. For example, LFA-1 is involved in antigen-dependent and antigen-independent interactions of immune cells. * Springer, T. Ao, 1987, Annual Review faun, 5: 223; Martz% E, 1986, Hum , Ian+unolo, 1:3° Most of the tilling is for LFA-1 LFA-1 was studied using monoclonal antibodies. binding to endothelial cells (Mentzer, S. J. et al., 1986, J. of C. e11. Ph 5io1. ．． 126:285), fibroblasts (Dustin, N. L. et al., 1986, J. of Imn+unol, 137: 245) , epidermal keratinocytes (Dustin, N. L. et al., 1986, J. of Ce11. Ph 5io1. ．． 107:321) and hepatocytes (Roos, E., and Roosien, F., 1987, Mu of S Cal, 1m. :553) has been shown to partially or completely inhibit the adhesion of T lymphocytes to I was attacked.

The role of MAC-1 in cell attachment was first demonstrated using monoclonal antibodies. It was proven using In such studies, MMC-1 was coated with C3b1 binding to erythrocytes, and such binding is associated with monochrome binding to MAC-1. was shown to be inhibited by null antibodies. Beller, B.1. et al., 19 82, J, of Ex, Med, 156: 1000゜Furthermore, MAC- 1 is Leishmania promastigotes (Leish+IlaniaProma) stigetes), Escherichia coli (E. coli) and Histoplasma cabusula Macrophas to zum()Iistoplasn+a Capsulatum) It was shown to be related to the binding of pages. Mo5ser, D, and Edels on, P., 1985. Mu of Immunol, 135: 2785 ; Wright, S., and Jong, M., 1986. J.

32-2 [-1-11 ≧ (21 ≧-2.-11 □ Ki to -5) Yu, 164,: 18T6; Bullock, W., and -rightA S., 1987 , J, of E Prisoner I, Mt.: 195, other studies have shown that MMC-1 is Chemotaxis of mesocytes and monocytes and their attachment to glass and plasti-7 surfaces. It has been shown to be involved in adhesion to monolayers of endothelial and epithelial cells.

p159.95 is associated with the adhesion of peripheral blood monocytes to the stroma and endothelial cells, elde, A. et al., 1987.7.61: 261, further p150. Studies using monoclonal antibodies directed against 95 have shown that it showed that it is utilized by cytotoxic T-lymphocytes in the formation of cytotoxic T lymphocytes.

The aforementioned studies, as well as additional studies, show that white blood cell integrins influence immune cell function. suggesting that it functions as a general adhesion protein for implementation. moreover, The aforementioned studies were directed against the α or β subunits of three integrins. Monoclonal antibodies were used. For the most part, studies have shown that the normal β subunit were shown to play a major role in the adhesion-related functions of these molecules. recently , cflN encoding human LFA-1, MMC-1 and p150.95 A clone was isolated. Kishimoto, T. et al., 1987, Dish, 4 8: 681 and Law, S., L. et al., 1987, EMBOJ,, 6 : 15-919.

As previously mentioned, inflammation is a significant part of an organism's defense against infection, and May cause extravascular tissue damage. Moreover, in some cases There is an uncontrolled inflammatory response, e.g. the response observed in septic shock. , which may contribute to the pathogenesis of the disease. White blood cells are at least partially Decomposes reactive oxygen metabolites, proteases and phospholipases at disease sites. By releasing acute ischemic shock, which causes damage related to was given. This is supported by research, in which peripheral blood Animals depleted of lymphocytes show significantly reduced damage from myocardial ischemia and reperfusion, Furthermore, reperfusion damage was induced by invivo administration of MAC-1 monoclonal antibodies. Minimum.

Finally, a rabbit model of hemorrhagic shock and resuscitation has demonstrated that the β subunits of MMC-1 Monoclonal antibodies against C11nical of liver and asteroid Invest, 81: 676 - Taken together, these results suggest that A number of disease states include myocardial infarction, hemorrhagic shock, and ischemia, such as and subsequent re-establishment of normal circulatory blood flow. leukocyte attachment via three leukocyte integrins in inhibiting damage to organisms and organisms. Reagents that block adhesion have significant therapeutic value.

Finally, ICAM-1, the endothelial cell receptor for integrin binding. It is worth noting that the receptor is also the receptor for rhinovirus binding. do. 5taunton SD, et al., 1990, Button, Dan: 243, Rhinowill. is a member of the picornavirus family and is responsible for about 50% of common colds. cause 5perber, S., and Hay-dern, F. (1988) Antimicorb, Agents Chesother, 32, vol. 409, p.

32. A preventive approach to prevent the common cold is to target cell-bound ICAM-1. The aim is to interfere with the binding of rhinoviruses. Actually, ICA? Solubility of 1-1 It was recently reported that a form of Marlin, S. D., et al., 19 90, Nature, 344: 70° In one plane, here The invention relates to a suitable membrane receptor for cells or virions (e.g., IC). undesirable transfer of cells or virions to cells or tissues expressing AM). harm and thus prevent or minimize diseases resulting from the binding of cells or virions. Describe the peptide that

A second aspect of the invention is to prevent undesirable binding of leukocytes or virions to tissue. inhibit leukocyte chemoattraction to tissues, thereby inhibiting leukocyte or To prevent or minimize diseases resulting from the binding of virions to On the other hand, the β subunit of leukocyte integrin, which competes with integrin, C Peptides with sequence homology to specific regions of D18 are described.

A third aspect of the invention is to inhibit leukocyte adhesion without affecting leukocyte chemotaxis. harm or prevent white blood cells from binding to tissue, thus preventing the unwanted binding of white blood cells to tissues and eventually white blood cells. A description of peptides that prevent or minimize disease to tissues resulting from the binding of blood cells. Ru.

A fourth aspect of the invention provides for leukocyte or virion cell adhesion and/or leukemia. A peptide that interferes with chemotaxis of the bulb, and can be used as a prophylactic or therapeutic agent for patients suffering from various diseases. is a description of a method of therapeutic treatment.

A fifth aspect of the invention provides peptides that interfere with leukocyte adhesion and/or chemotaxis. and suffer from various illnesses, especially those caused by rhinovirus infections. This is a description of a method for prophylactically or therapeutically treating patients suffering from cancer.

A sixth aspect of the invention provides antibodies against (:D18 peptide, and prophylactic use of antibodies). and description of therapeutic applications.

Yet another aspect of the invention is the description of peptides that inhibit the cell adhesive properties of leukocytes. , where the peptide is not easily hydrolyzed and thus the extended tnvi It shows the circulation time of vo.

These and other aspects of the invention will become apparent upon a thorough consideration of the invention described below. Will.

Figure 1 shows the amino acid sequence of CD18, a β subunit of leukocyte integrin. show. The underlined region is synthesized and active in the leukocyte/endothelial cell adhesion assay. Corresponds to the peptides tested for sex.

We also synthesized smaller peptides within a region and tested them for activity. . Each peptide is designated by a number in the drawing, i.e. 1-6, and a second number means the number of amino acids in the peptide. Thus, 1-26 is 26 amino acids It means a peptide with a region l. Region 1 consists of only 26 amino acids. 1-26 means a peptide spanning the entire region. However, 1-1 5 means a peptide from region 1 consisting of 15 amino acids. This amount of peptide The child is numbered from the carboxyl to the amino terminus.

Table 1 Tested for the ability of pleomorphic nuclei to inhibit leukocyte adhesion to endothelial cells , shows the amino acid sequences of various CD18 peptides.

Table 2 shows the effect of polymorphic nuclei on leukocyte adhesion over a concentration range of 10-4 to 10-''M. The inhibitory effects of some CD18 peptides are shown. 4-49 at 10-'M It is clear that it is most effective.

Table 3 uses polymorphonuclear leukocytes from different human donors and polymorphism over a concentration range of 10-4 to 10-''M after storage under various conditions. Figure 4 shows the effect of peptide 4-29 on nuclear leukocyte adhesion.

Table 4 shows the polymorphisms from different donors over the concentration range from 10-4 to 10-'M. Effect of peptide 4-15 on polymorphonuclear leukocyte adhesion using polymorphonuclear leukocytes. Indicates purpose.

Table 5 shows CD18 peptide, -26,2-24,3-29,4-29,5-2 4 and 6-25.

Table 6 shows the chemical qualitative inhibitory activity of C[118 peptide, 4-29.

The invention described herein takes advantage of previously published work. As an example, like this Research consists of scientific papers, patents or pending patent applications. Quoted above and below All of these publications and applications are incorporated herein by reference.

The present invention consists of several unique methods and compositions. Aspects of the invention can be found here will be explained separately.

Various diseases, preferably those resulting from troublesome adhesion of leukocytes or rhinoviruses Cell-cell or cell-virion adhesion, which is a useful drug for the treatment of Qi. Peptides have been discovered that prevent or interfere. These peptides stimulate leukocyte intelligence. Amino acid sequence homologous to a region of the beta subunit of Glin, CD18 has.

Peptides incorporating the following 5 amino acids are preferred: NH2-Trp-Arg-A sn-Val-Thr-Arg-C008 Among large peptides with the following sequence: More preferred are the above peptides incorporated into: More preferred is the above peptide incorporated into a larger peptide with the following sequence: stomach: NH2-De-G “y-Trp-Arg-^1n-VaJ-’r’hr-Arg -Lcu-Leu, Val-Phe-Alx-Thr-As blowing|COOH Figure 1 shows the amino acid sequence of the beta subunit of leukocyte integrin, CD18. columns are shown and peptides that interfere with leukocyte or virion attachment are underlined. It's dark. Learn more about the ability to compete with peptides and activated endothelial cells was tested for its ability to prevent leukocyte adhesion. In addition, leukocyte chemotaxis The ability of these peptides to inhibit was determined.

The aforementioned peptides can be prepared using techniques well known in the art, e.g. ence, 232: 341-347 (1985). It can be produced by Merrifield's solid phase method. This step is a commercially available synthesizer, e.g. Biosearch The 9500 automated peptide instrument can be used to cleave blocked amino acids. Determination was achieved using hydrogen fluoride, and the peptide was purified by preparative HPLC. Waters Delta Prep 30 Using a 00 instrument, 15-20 μm Vydac C4 Prev. Purify with PrepPAK.

As one of ordinary skill in the art will appreciate, the exact chemical structure of the preferred CD18 peptide is not disclosed herein. Although the specific changes to the structure may be desirable depending on a number of factors, There are some bad things. For example, the peptide may be acidic or can be incorporated as a basic salt or in neutral form. In addition, The primary amino acid sequence of proteins is modified by derivatization using sugar moieties (glycosylation). or other auxiliary molecules such as lipids, phosphates, acetyl groups, etc. and 7-carido, polyethylene glycol (PEG) and polio It can be increased by conjugation with xyethylene glycol (POG). child Modifications such as included in the definition of peptide in Of course, such modifications can be activity by enhancing or decreasing the activity of the protein in the assay. It is expected to have a quantitative or qualitative impact. Furthermore, especially those skilled in the art It is important to understand that peptide drugs with increased inviν0 residence time are may be advantageous in certain applications. The in vivo residence time of the peptide is can be increased using methods known in this field, and two An exemplary method for synthesizing peptides having substantially non-hydrolyzable peptide bonds or a polymer that binds to or associates with a biocompatible polymer. This includes synthesizing peptides. Examples of polymers are [l1brich, K, et al., 1986, Ha material 9 Seki LCh eaa, 187: 1131; and R ihova, B. et al., 1986, J-, Ogc Bandaiyama” Nito I, Koasa: 221 ,It is described in.

As one of ordinary skill in the art will appreciate, the peptides described herein are readily available to animals, including humans. Can be administered alone or in combination with other anti-inflammatory agents, or may be combined with a pharmaceutically acceptable diluent or carrier. This way are well known to those skilled in the art and can be formulated according to standard pharmaceutical practice. It will be done.

Examples of diluents are physiological saline or buffered saline, as well as Ringer's solution. liquid and dextrose injection, and dextrose saline and lactic acid Ringer's Injection or Additional Therapeutic Agents, Preferred in the Treatment of Inflammatory Conditions Includes antibiotics or antibodies known to be effective.

Leukocyte adhesion is measured using several assays known in the art. and a preferred assay is Char.

et al., 1985, Tandotaru, 65: 473. Simply put, This assay involves labeling leukocytes with an appropriate label and incubating the leukocytes with endothelial cells. tion and determining the number of adherent leukocytes. Preferably thin The cells are labeled with a gamma-emitting isotope, and the preferred label is I11. 51 chromium.

Leukocytes can be obtained from human donors using standard techniques. Generally, this This separates the blood into a physiologically balanced salt solution containing a suitable anticoagulant. , and by a suitable separation step, preferably Ficoll-Heiberg (F i co　　　　　　-Hypaque) gradient. contaminating red blood The spheres can be removed by hypotonic lysis. Physiologically buffers the resulting white blood cells suspension in a solution, pH 7.4. Preferred physiologically buffered solutions are Bank's balanced salt solution without lucium and magnesium.

The isolated leukocytes are then exposed to the desired radioactive isotope for a suitable period of time, typically 15 minutes. Labeling is performed by incubation at a predetermined concentration. Ru. The radiolabeled cells were washed to remove unincorporated label, and then The attachment assay described below is carried out by suspending in an appropriate solution.

Endothelial cells can be obtained from a number of sources and by several techniques known in the field. It can be prepared from Preferably, the endothelial cells are obtained from human sea bream veins with Ch. obtained using the procedure of aro et al., supra. In general, endothelial cells are By elementary digestion, preferably Jaffe, E. A. et al., 1973, J. of Collage as described in Cl1n, Invest, 52:2745. Isolate using genease. Cells are placed in a suitable tissue culture sublayer, preferably gelatin. Grows on coated surfaces.

Endothelial cells can be grown in a variety of tissue media, and such media may be auxiliary substances, such as fetal calf serum at appropriate concentrations, and those routinely available to those skilled in the art. Other auxiliary substances/additives recognized as suitable for the endothelial cells being used Contains. Endothelial cells receive appropriate proteases and, if necessary, metal ions. Can be subcultured in dilute solutions of chelating agents. Preferably 0.05~ Use a solution containing 0.25% trypsin and 0.02% EDTA . Cells were characterized by immunofluorescence to ensure that the cells were in fact endothelial cells. Test for child III antigen, a known endothelial cell marker.

Adhesion of leukocytes to endothelial cells can be determined as follows. Generally the fifth Beyond subculture, early-passage endothelial cells are grown on a suitable support and in a suitable Culture in cell culture medium. The culture support preferably promotes endothelial cell attachment. Pre-coat with a suitable reinforcing substance. Some such substances, e.g. Bronectin, poly-L-lysine, gelatin and lamin are known. Fi Bronectin is preferred. A suitable culture support is a 96-well microtiter plate. and suitable media include fetal calf serum and well known to those skilled in the art. and other supplements known to be beneficial for endothelial cell proliferation and maintenance. This is a medium 199 containing auxiliary substances. Addition of a predetermined number of labeled leukocytes Physiologically normal serum containing a reduced amount of fetal calf serum, preferably 1%. Wash the endothelial cell monolayer with equilibrated salt solution. The preferred solution is 1% fetal calf RPMI supplemented with serum.

The monolayer of endothelial cells containing added leukocytes was inflated to allow maximum attachment of leukocytes. incubate for a period of time, preferably at 37°C. The test is carried out for 30 minutes in a suitable cell culture atmosphere. Generally, this will keep the cells at 5% CO2,9 Grow and incubate for the duration of the assay in 5% air and 95% humidity. It consists of

The unadherent leukocytes are then removed by a number of techniques known in the art. by measuring the amount of radioisotope associated with a monolayer of endothelial cells. The number of leukocytes adhering to the monolayer of endothelial cells is then determined. Fundamental connections, i.e. , a control is performed to account for binding to endothelial cells that are not activated with TNF.

In a typical experiment performed in four replicates, the assay was highly reliable. gives a standard deviation of less than 10% of the mean, usually less than 5%. typically , the results were added to endothelial cells that remained attached after removing non-adherent cells. Expressed as a percentage of white blood cells.

Using the above assay, typically the peptide to be tested is concentrated over a range of concentrations, preferably Preferably, it is added over a range of 10-'M to 10'M. As mentioned above, the peptide synthesized, lyophilized, and dissolved in 15-25 μm dimethyl sulfoxide. This volume is then transferred to a suitable medium, e.g. containing 1% fetal calf serum. Ru! ? Dilute appropriately in PM I to the desired final concentration to be tested.

Endothelial cells were incubated in RPMI with 1% fetal calf serum for 2 hours before addition of leukocytes. At least 4 hours at 125 U/mg of TNF with a specific activity of XIO'07 mg Activate for a while. TNF is a receptor for the binding of integrins on leukocytes. inducing the expression of ICAM on the surface of endothelial cells.

Materials and methods that establish the chemotactic-inhibiting properties of peptides are generally in the field. A known and preferred procedure is Capsoni et al., 1989, J. of Ismunol, Meth, 120:125. one In general, chemotaxis involves placing leukocytes and chemotactic substances on opposite sides of a membrane in a suitable medium. Determine by placing. The preferred device for performing chemotaxis assays is Costar Corporation (Costar Corporation) Cambridge, S.C.) and Trans-Well It is called a culture device. So that white blood cells do not have unrestricted access to substances, Select membrane size; rather, if a chemotactic response is elicited, leukocytes It attaches to and moves into and through the filter. Inhibitory activity against a certain substance If you are testing for This can be achieved by

The procedure of Capsoni et al., supra, was used with the following modifications. White blood cells are on isolated and labeled with +1 indium as described for the attachment assay. can be done. Approximately 5X10' cells/+ml in appropriate cell culture medium after labeling the cells. Resuspend with Next, the inhibitory activity of the determined amount is determined by taking a desired amount of cell suspension in advance. one or more peptides to be tested, and this mixture is Add to device. Place the 3 μm pore membrane in a 24 trans-well tissue culture plate. Place it in Incubate the cell/inhibitory peptide mixture for a short period of time at 37°C. The cells are incubated to adapt to the membrane surface, and then the cells settle to the membrane surface. Give yourself enough time to Next, insert into wells containing cell culture medium. cents. This medium contains thymosan-activated human serum at approximately 0.5%.

Activation of thymosan is a complement-induced chemotactic agent that attracts leukocytes through membrane pores. Generate a child. This medium may also be used for an appropriate period of time before addition to cell culture wells. Prewarm for a while. After 30 min incubation at 37°C, CD1 8 The number of leukocytes that migrated through the membrane filter in the presence of the peptide into the medium It is easily determined by counting the amount of 1st indium present. this is , add appropriate detergent to the wells at the appropriate concentration before removing aliquots for counting. can be promoted by adding it to the medium in the medium. In this way, try The inhibitory activity of the peptide under investigation can be determined.

Effect of CD18 peptide on rhinovirus binding Abraham, G.

and Colonno, R.J., (1984) J. Virology, 51 , p. 340, Marlin, S. D. et al. (1990) Natur e, vol. 344, p. 70. and can be determined using materials. Briefly, this procedure media containing appropriate radioisotopes by methods known in the art. grow virus-infected cells in a culture medium and isolate the virus from the culture medium The method consists of radiolabeling the rhinovirus. polyethylene Virions were precipitated from the medium using Recall, then 30% sucrose. It is particularly effective to pelletize the villion with a stepwise gradient of .

Radiolabeled virions express ICAM-1 in cell adhesion assays. It can be used by using cells that can be used. Preferred cells are as described above. or the endothelial cells prepared by Abraham, G., and Co. 1onno, Rj.

This assay is essentially as described for media of neutrophil attachment to endothelial cells. This can be carried out by adding the appropriate CD18 peptide to the assay mixture. Ru. Peptides that bind to ICAM-1 and prevent virion binding are By counting the number of virions that bind to a monolayer of cells in the presence of putido Easily identified will be verified.

Antibodies, polyclonal or monoclonal or recombinant, the latter preferably (has been humanized) can be generated for inhibitory peptides. this Such antibodies are used to bind to the CD18 peptide present on leukocytes, This will disrupt or prevent leukocyte adhesion to the endothelium.

Monoclonal antibodies were prepared by Kohler, G., and Milstein, C. 1975, Nature, γ56:495 (which is not known in the field). using the general procedure described in , can be generated. This initial study demonstrated that murine lymphocytes and drug-selectable This included fusion of different plasmacytomas to generate hybridomas. Continuing This technology can be used to produce hybrid cell lines that secrete human monoclonal antibodies. Applied. The latter procedure is generally described in Abrams, P., 1986, Met Hod mutual” Dan u! 121:107, but other changes are here. known in the field of

Antibody-secreting cells, whether murine or human, produce antibodies. Combine with a fusion partner and fuse the cells by electrofusion or with a suitable fusion agent. , preferred polyethylene glycol, more preferably polyethylene glycol 1 Fuse using 000.

Gently stir the latter into the cell pellet containing the antibody-secreting cells and fusion partner. Add in small amounts over a short period of time. After addition of the fusion agent, the cell mixture Wash to remove fusion agent and cell debris, and remove fused and unfused cells. Place the cell mixture consisting of the cells in a suitable cell culture chamber containing a selective growth medium. - inoculate into. After a period of 1-3 weeks, the cells become evident and start producing antibodies. identification and subcloning to obtain stable monoclonal cell lines. Ensure possibility.

Preferred antibodies are human monoclonal antibodies, which are peptide-mediated in vivo. or antibody-producing hybrid cell lines from in vitro sensitized lymphocytes. cells, thereby creating an available permanent source of the desired antibody. Therefore, it can be produced. inviv.

Although the technique of permanent mitosis is well known in this field, 1nvitro The technique is generally described by Luben SR, and Ohler, M., 1980. old cr obiolImmuno1. ．． 17: 635, Reading, C,, Methods in Enz + l0I0, 121 (Part 1): 18, also Voss, B., 1986, ods in Enz 5olo■, 12 1:27. A number of in vitro immunization systems are available for human B. It was shown to be effective for sensitizing cells. Reading, C, 198 2, J, of Tmmunol, Methods, 53: 261° sensitization The infected lymphocytes can become permanently mitotic by infection with a virus. Hitlin The preferred viral transformation technique for Pakocytes is the Epstein-Barr-Will This includes the use of This virus is able to transform human B cells and has been used to generate human monoclonal antibodies. Crawford, ozbor in D. et al. 1983, J. Gen. Virol, 64:697; , V., and Roder, J. 1983, J.

Button Shikasan iron grain, ↓Near 2゜ Another procedure for permanently rendering sensitized lymphocytes is a combination of the above two techniques. consists of viral transformation and cell fusion. preferred combination For this purpose, antibody-secreting cells were transformed with Epstein-Barr virus and subsequently It consists of fusing the transformed cells with a suitable fusion partner. The fusion partner is Mouse myeloma cell line, heteromyeloma line, or human myeloma line, or other permanent cell line. It can be a cell line that has been rendered functional. PCT Patent Issue No. 8110 0957; Schlom et al., 1980, PNAS USA, 77: 6 841; Croce et al., 1980, Nature, 288: 488 ゜A preferred fusion partner is a mouse-human heterohybrid, more preferably a mouse-human heterohybrid. The cell line is designated as F3B6.

This cell line is available from the American Type Culture Collection (America). n　Type　Culture　Colection) with acceptance number 11B8 It was commissioned in 785. It was commissioned on April 18, 1985. F3B6 The procedure that occurs is described in European Patent Application No. 174,204. eps Use of Tine-Barr Virus Transformation and Permanent Antibody-Producing Cell Lines The technology applicable to the production of is described in Roder, J. et al., 1986, Methods In Enz 5olo Ou, 121: 104. Basically, This procedure uses Epstein-Barr virus as a suitable source, generally an infected cell line. The target antibody-producing cells are then isolated from the virus-containing supernatant. Ru. Cells are washed and cultured in appropriate cell culture medium. Subsequently, cell culture Epstein-Perwy The transformed antibody-producing cells are identified by the presence of antigen in the nucleus of the virus, and the transformed antibody-producing cells are standard can be identified by methods known in the art.

Antibodies to the peptide can be prepared using a suitable carrier, as is known in the art. produced by standard coupling procedures to molecules of Yer. preferred procedure and The composition is disclosed in U.S. Pat. No. 4,762,706. For example, the antibody can be used in a host animal, e.g. Injecting peptides or peptide fragments into herons, rats, goats, mice, etc. produced by. Prior to injection, the peptide is first transferred to a suitable carrier molecule, preferably or keyhole limpet hemocyanin (KLH) or bovine serum albumin ( BSA). The conjugation is preferably between cysteine residues on the peptide. This is accomplished via the fuhydryl group. If the peptide lacks a cysteine residue, It can be added using known methods.

Hetero-heterobifunctional cross-linking reagents are used to link carrier proteins and desired peptides can be coupled. A preferred coupling agent is 1-hydrocarbon. N-Maleimi of droxy-2-nitro-benzene-4-sulfonic acid sodium salt do-6-aminocaproyl ester, and its preparation and use are in this section. known in the field and described in U.S. Pat. No. 4,762,706. There is.

Having described what Applicants believe to be an invention, the following examples are considered to be examples of the invention. These examples are provided for illustrative purposes only, and these examples should not be construed as limiting the scope of the invention. For example, changes in the source, type, or method of producing the antibody; different labels and and/or signals; different materials and configurations of test supports; different immunization methods. may be used without departing from the scope of the invention.

Synthesize the peptides shown in Table 1 and inject polymorphonuclear leukocytes into monolayers of human endothelial cells. Tested for ability to interfere with or block adhesion. These pep Chido corresponds to the underlined structure of CD1B shown in Figure 1. . Smaller peptides within a region can also be synthesized and tested for activity. Ta.

Each peptide is designated by a number in the drawings, i.e. 1-6, and the second number is the peptide of that peptide. Refers to the number of amino acids in the peptide. Thus, 1-26 also contains 26 amino acids. It means a peptide with two regions. Since region 1 consists of only 26 amino acids, 1-26 means a peptide spanning the entire region. However, 1-15 means a peptide from region 1 consisting of 15 amino acids. Peptide is the molecule They are numbered from the carboxy terminus to the amino terminus.

Peptides were synthesized using Merrifield's solid phase method. Merrif 1eld, R,B,, 1963, J,of Am,Chem,Soc,, 75 : 48-79, Bio-search 9500 automated page A peptide device was used with T-Boc amine protection. Cut using hydrogen fluoride and the resulting peptides were analyzed by preparative high performance liquid chromatography. Waters Deltaprep 300 0 with a PrePak CI8 column to aqueous acetonyl Purified using tolyl-trifluoroacetic acid (TFA) mobile phase.

l engineering The peptides were lyophilized and stored dry at 4°C. Activates peptides When testing a sample, weigh it and place it in an Eppendorf tube. and dissolved in about 15-25 μm dimethyl sulfoxide, then 250 μl with assay medium consisting of RPMI containing 1% fetal calf serum. Dilution gave a 4 x 10-'M source solution. If necessary, these source solutions An aliquot was taken and stored at -70°C. Before performing the assay At 104.10-5.1, 4X10-'M source solution was added to the tube in the assay. 0-6.10' and diluted to a final concentration of 101M.

Endothelial cells were isolated from human lung zones by mild collagenase digestion.

Collagenase is manufactured by Worthington Co. Freehall, New Jersey), and The general procedure is described in Jaffe, E. A. et al., 1973, J. of Cl1n, In. Best, 52:2745. Obtained from collagenase digestion Place the cells on a gelatin-coated flask in 25ml of Hepes-buffered medium 1. 99 (Gibco, Grand Island, NY). It grew inside. This medium was supplemented with 20% fetal calf serum. Also, this culture The background was 60 μg/ml sodium heparin (Sigma Corpora-ti St. Louis, MS), 2mM l,-glutamine and 50μg /ml of bovine hypothalamus extract. Bovine tissue was prepared using Bel Freeze (PalF). Hypothalamic extract (obtained from A. Reeze) (Lougarth, Alaska), hypothalamic extract Acts as a growth factor for skin cells. The pH of the cell culture medium was 7.4.

After the endothelial cells reach confluency, they were incubated with 0.02% EDTA. subculture in 0.25% trypsin and subsequently using the same solution. Secondary culture was performed. Bank cells into this mixture in equilibrated salt solution at room temperature. Exposure was for approximately 1 minute.

Finally, seed Habo2XIO' cells/well into microtiter plates. Ta. The nature of the endothelial cells is determined by their cobblestone morphology and their confluency. The fact that they stain positive for factor VIII antigen by indirect immunofluorescence confirmed by both. The latter procedure is well known in the field and Jaffe E, ^9.1973, J, Cl1n-1nvest, Sz: 2745.

Monolayers of endothelial cells were incubated with 20% fetal calf serum, 25% prior to the 5th passage. Medium 199 containing +sM Hepes, pH 7.4, and other supplements as mentioned above. Inside a polystyrene 96-well flat-bottomed microtiter plate ( Cornig Corporation). micro titer pre The surface of the sheet was treated with 6.4 ng/l of human plasma fibronectin at 25°C. After incubation for 30 minutes, endothelial cells were plated.

Cultures of endothelial cells were used when they were confluent. Inside Skin monolayers were washed with RP)'II + 1% fetal calf serum and 125U/ml activated with TNF and then labeled with polymorphonuclear leukocytes and 5X10' cells/well. were incubated at a final concentration of . Pellet cells onto monolayer of endothelial cells for 30 min I let it happen.

Human polymorphonuclear leukocytes were collected from adult volunteers from cicadas and treated with anti-I1 solids (10 % heparin) and then Ficoll-Hyberg The blood was obtained by centrifugation on a Paque) gradient.

Contaminating red blood cells were removed by hypotonic lysis. The remaining cell population is 95-98% consists of polymorphonuclear leukocytes, and these cells are stored in a bank of balanced salt solutions p[+7 ．． The cells were suspended at a concentration of 5 oxio'' cells/1 cell in 4 ml.

■1 Indium oxide (100u Ci/10"PMN) for polymorphonuclear leukocytes (10aiCi/v++1, (Amersha* Corporation ). Labeling was carried out for 15 minutes in bank solution at room temperature and then The labeled cells were isolated by centrifugation for 5 minutes and the remaining unincorporated cells were isolated by centrifugation for 5 minutes. To remove the labeled label, wash twice with Bank's balanced salt solution, then wash with 1% Suspended in RPM+ supplemented with calf serum.

As described above, 5X10'/well of labeled Pl'IN cells were transferred to 96 wells. into a microtiter plate. Incubation at 37°C The culture was carried out in an atmosphere of 5% CO, 95% air in a 11-culture incubator. provided.

After 30 minutes of incubation, during which time polymorphonuclear leukocytes have formed into a monolayer of endothelial cells. and fill the microtiter plate with adhesive clear plastic. (Dynatech, Inc., Alexander, VA). Handstand, and Beckman Instruments Corporation (Bec Microprop obtained from kman In5true+ents Corp. Centrifugation was performed using a rate carrier. Centrifuge at 75 x g for 5 minutes at room temperature. It was carried out for minutes. This effectively removed non-fouling material. , then blotte the plate polymorphonuclear leukocytes that remain attached to a monolayer of endothelial cells. The number of was determined using a gamma counter. The results are shown in Table 2, and they is expressed as the percentage of polymorphonuclear leukocytes that remain attached to the endothelial cell monolayer. There is.

The data presented in Table 2 are suggestive in several respects. First, the most active Petide is 4-29. That is, the fourth peptide has 29 amino acids. Derived from D18. This peptide showed significant inhibition of PMN adhesion. This molecule exhibits peak activity in the range of 10-5 to 10-bM. In contrast, the fourth peptide, a 14-amino acid peptide, shows almost no activity.29 The range of activity for 7- varies over the concentrations tested.

deep red PMN to TNF of CD18 peptide % change in average Peptide 10-'M 10-'M 10-'M, 10-'M 10-'M available A significant number of peptides showed activity at 10-'M, but at lower concentrations. showed little or no activity, and none of these peptides showed 1 4-29 was not active over the range of 0-5 to 10"M. CD1 Peptide 1-26) of 26 amino acids of the first peptide of 8 is 10-4 to 10-' Similarly, the CD18 phase showed inhibitory activity with a clear peak in the range of M. 2 24 amino acid peptide (2-24) in the tiI region, and 3-29 is a 10- The peptide that showed activity in 'M and showed almost no inhibitory activity was this molecule. and 14 or 24, respectively. or contains 16 or 25 amino acids.

Finally, the JUN- and -T peptides are polymorphic in the concentration range of 10-'M. Peptide 2X was used at a concentration of 10-4-10-''M to inhibit leukocyte adhesion. It was observed to have activity over a range. Tags containing these peptides Proteins are known in the art, or as described by Angel, P. et al. 1988, Nature, 332: 166.

Polymorphonuclear leukocytes from different individuals determine the efficacy of peptides in adhesion assays. studies using peptide 4-29 with the intention of determining whether it has any A study was conducted. We also determined the stability of the peptide to various storage conditions. Conclusion The results are shown in Table III.

In experiments using polymorphonuclear leukocytes isolated from donors 100 and 272. demonstrated that freezing and thawing the peptide does not significantly affect its activity. Ta. Moreover, the activity of the peptide is dependent on the donor from which the polymorphonuclear leukocytes were isolated. There are fluctuations in opinion. Finally, as Table III also shows, the activity of the peptide is 4 ℃ for different periods of time after storage.

l lord Only CD18 peptide #-29 m- of PAN to TNF. ・・Water endothelial cells were activated with IL-1.

with the intention of determining whether shorter peptides within this region have activity. , 4-29. 4-14ha was mostly active. 4-15 was tested and the results are shown in Table 4. clear As shown, 4-15 is active, and the peak of activity is in the range of 10-4 to 10-'M. It is in. Variability in the assay was observed as a function of the donor from which the PMNs were isolated. It was.

l soil To seal 1ヱIUη Donor 10-'M 10-'M 10-'M average -58±7 -41±8 -17±2 donors 10-'M 10-'M 10-'M average -24±10 -5±3　-6±2　Jul Engineering / CD18 peptide to polymorphonuclear leukocyte chemotactic response to 0 blood Many of the materials and methods used to test the inhibitory activity of CD18 peptides , similar to that used to perform the adhesion assay described in Example 1. , or identical to them. Isolate polymorphonuclear leukocytes as previously described and and labeled with 1' indium, and the cells were incubated in RPM11640 medium with 5XI. It was suspended at a concentration of O'/ml. Next, 75 μl of cell suspension and 25 μl of Add medium containing the desired concentration of CD18 peptide to the trans-well inserts. and the mixture was ink-baked for 10 minutes at 37°C. .

Add 0.6 ml of 0.5% zymosan activation to the bottom well of a 24-well plate. Medium containing human serum was added. The solution was also heated to 37°C for 10 minutes before use. It was heated for a while.

Next, trans-wells containing the cell suspension with the desired CD18 peptide Incubate the inserts for 30 minutes at 37°C in a 24-well plate. The assay was performed by

Subsequently, remove the insert and add 60 μl of 10% sodium dodecyl sulfate. The solution was added to the wells of a 24-well plate. Calm the plate Incubate for 15 minutes at room temperature with shaking and add 110 μl of Remove the aliquot and determine the amount of indium with a gamma counter. did.

The results are shown in Table 5, which shows that the peptide Expressed as % inhibition or enhancement of migration caused by Data is 4 people isolated from different human donors No. 591.593.195 and 499. were collected from polymorphonuclear leukocytes. As is immediately apparent when looking at the data, the peptide Concentration range from 10-4 to 10-5 M for 2-24.3-29 and 4-29 There is significant inhibition of chemotactic migration within the range. The greatest inhibition was It is a function of both the concentration of putide as well as the donor from which the polymorphonuclear leukocytes were isolated.

Table E Peptide 591 593 195 499 *Data shows migration of polymorphonuclear leukocytes Expressed as % inhibition (-) or enhancement.

Due to variations in results observed using polymorphonuclear leukocytes from different individuals Additional experiments were performed to confirm the inhibitory effect of the most active peptide 4-29. did. The experiment was performed using polymorphonuclear leukocytes from six donors and 195 was used here and in previous experiments. Furthermore, experiment 1 A concentration range of 0-4 to 10-7 was performed. The inhibitory activity of this peptide is confirmed. demonstrated and significant activity was observed for all donors. The peak of activity is 10 -'M. Polymorphisms from different donors as examined in previous experiments It should be noted that there is a variable percentage of inhibition using nuclear leukocytes.

deep red Effect of CD18 peptide on neutrophil chemotaxis Peptide concentration Cells migrated through the filter in response to 10.5% thymosan-activated human serum. Measured by total number of cells.

1 serving 1 to - inovirus people Using the assay described in Example 1 and varying amounts in the assay mixture of peptide and about 1X10' counts/min. , the ability of peptide 4-29 to inhibit rhinovirus binding to activated endothelial cells. was tested. Radiolabeled rhinovirus was produced by Abrahas, G. and Colonno, R.J.

can be generated. Pre-ink the endothelial cells for 30 min at 4-29°C. conjugation to prevent endocytosis of the peptide. Approximately 50-100 A concentration of tt g/ml provides approximately 50% inhibition of rhinovirus binding. It will be determined that.

1" eye ・CD18 peptide for use as a drug The effects of the common cold can be reduced by incorporating an effective amount into an acceptable nasal carrier. Can be applied for effective prevention. This amount is determined experimentally by one skilled in the art. but generally will be in the range of about 50 μg to Img/wl. nasal pharmaceutical formulations Well known to those skilled in the art, and from Remington's Phar+ma Ceutical Sciences”, 14th edition, 1970. . However, it will be appreciated that selection of the appropriate carrier will depend on the specific nasal administration desired. It will depend on the nature of the form. i.e. peptides as drops or spray Formulated as a nasal solution, nasal suspension, nasal ointment, or nasal gel for use where peptide 4-29 is placed in a physiologically compatible solution. exist. This solution also contains small amounts of emulsifying or dispersing agents, buffering agents, preservatives, humectants, etc. Wetting agents and gelling agents may be included as known in the art. Ru.

Peptide 4-29 in the form of a nasal spray can be used to prevent individuals from being exposed to other individuals who have a cold. It can be applied to individuals before or immediately after exposure. in this way Therefore, peptide 4-29 may prevent or reduce the amount of rhinovirus binding to the nasal endothelium. The exposure of individuals to rhinoviruses by infected individuals is minimal as it is greatly reduced. or can be eliminated.

The invention has been described with reference to specific embodiments. However, this application Changes that may be made without departing from the spirit and scope of the claimed claims and Intended to encompass changes.

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Procedural amendment (formality) %formula% 1.Display of the incident PCT/US 91106053 2. Name of the invention Peptide drugs for disease treatment 3. Person who makes corrections Relationship to the incident: Patent applicant Name: Sitas Oncology Corporation 4, Agent Address: Shizuko Toranomon Building, No. 8-10, Toranomon-chome, Minato-ku, Tokyo 105 Phone: 350 4-07215, date of amendment order 6. Subject of correction (1) Certificate of change of patent applicant name (2) Translation of the description, claims and abstract 7, contents of amendments (1) As per attached sheet (2) Translations of the description, claims, and abstract (no change in content) 8. List of attached documents (1) Certificate of change of patent applicant name and one copy each of its translations (2) Specification, scope of claims, and abstract: one translation each However, regarding (1), please refer to the August 1992 attachment and attach a copy. I will attach it.

international search report lIl+ww*wmalAmla+++e*Ns Dr Ding/IIζ 01/l’ 1ln('1Pffm PCT/1W210 +s+mmw+m lI'1ML t211・PI41211111fl Front page continuation (72) Inventor Mundi, Kirsten United States of America, California 94 553゜Martinez, Heather Leaf Lane

Claims (1)

[Claims] 1. CD18, a leukocyte beta subunit that prevents leukocytes from adhering to biological materials. , integrin peptide. 2. The peptide is a) [There is an array] b) [There is an array] c) [There is an array] d) [There is an array] e) [There is an array] f) [There is an array] A peptide according to claim 1 selected from the group consisting of. 3. The peptide according to claim 2, wherein the peptide consists of [sequence] Petite. 4. The peptide is [There is an array] The peptide according to claim 2, which consists of a peptide comprising: 5. The peptide is [There is an array] The peptide according to claim 2, which consists of a peptide comprising: 6. The peptide is [There is an array] The peptide according to claim 2, which consists of a peptide comprising: 7. The peptide is [There is an array] The peptide according to claim 2, which consists of a peptide comprising: 8. The peptide is [There is an array] The peptide according to claim 2, which consists of a peptide comprising: 9. Claims characterized in that the biological material has an ICAM The peptide according to item 1 below. 10. The peptide according to claim 9, wherein the biological material is endothelium. 11. Interfere with or prevent the binding of leukocytes to biological materials and having the property of interfering with or preventing the chemotaxis of blood cells to biological substances , leukocyte β subunit, CD18, integrin peptide. 12. Leukocyte β, which interferes with or prevents the attachment of leukocytes to biological materials. Effective amount of one or more subunits, CD18, and integrin peptides A method of treating a disease comprising administering to a subject suffering from the disease. 13. Treating the disease according to claim 12, wherein the disease involves an inflammatory response. How to do it. 14, the leukocyte β subunit, CD18, and integrin peptide are a) [ There is an array】 b) [There is an array] c) [There is an array] d) [There is an array] e) [There is an array] f) [There is an array] 14. The method of claim 13, wherein the method is selected from the group consisting of: 15, the leukocyte β subunit, CD18, and integrin peptide have the following sequence: there is】 A method for treating the disease according to claim 14, comprising: 16. Claim 12, wherein the disease is caused by a rhinovirus. How to treat the listed diseases. 17, antibodies against leukocyte β subunit, CD18, and integrin peptides. 18, a) [There is an array] b) [There is an array] c) [There is an array] d) [There is an array] e) [There is an array] f) [There is an array] The effectiveness of one or more antibodies binding to a peptide selected from the group consisting of A method of treating a disease comprising administering an amount to a living subject. 19. [There is an array] treatment of a disease, which consists of administering to a living subject an effective amount of an antibody that binds to a How to put it. 20. Claim 18, wherein the disease is caused by a rhinovirus. How to treat the listed diseases. 21. Claim 19, wherein the disease is caused by a rhinovirus. How to treat the listed diseases. Biology of leukocytes selected from the group consisting of 22, HT, JUN-k or 2X Peptides that interfere with adhesion to target substances.